Antibody responses to BNT162b2 SARS‐CoV‐2 mRNA vaccine among healthcare workers and residents of long‐term care facilities: A cohort study in Northern Italy

Abstract Background and Aims Long‐term care facilities (LTCFs) have been severely impacted by COVID‐19, with a disproportionate amount of SARS‐CoV‐2 infections and related deaths occurring among residents. Methods This study is part of an ongoing multicenter, prospective cohort study conducted among healthcare workers (HCWs) and residents of 13 LTCFs in Northern Italy designed to evaluate SARS‐CoV‐2 specific immunoglobulin class G (IgG) titers before and following vaccination with Pfizer/BNT162b2 SARS‐CoV‐2 mRNA vaccine (two doses of vaccine, 21 days apart). Serum samples were obtained from participants (t0) before vaccination, and (t1) 2 weeks after and analyzed to determine anti‐S1 IgG antibodies. Results Five hundred and thirty‐four participants were enrolled (404 subjects participated in both blood draws). Seropositivity was 50.19% at t0 and 99% at t1, with a significant difference in IgG titers. A higher proportion of residents were seropositive at t0 compared with HCWs, with significantly higher IgG titers among residents at both t0 and t1. Pre‐existing immunity also had a significant effect on postvaccination IgG titers. However, a significant difference in titers at t1 between HCWs and residents considering only participants seropositive at t0 was found, with higher median titers among previously seropositive residents. Conclusion Findings of this study provide scientific evidence endorsing the policy of universal vaccination in LTCFs.


| INTRODUCTION
Long-term care facilities (LTCFs) residents represent a high-risk population in a high-risk setting for SARS-CoV-2 transmission. 1 LTCFs have been severely impacted by COVID-19, with a disproportionate amount of SARS-CoV-2 infections and related deaths occurring among residents in several countries, and in Northern Italy in particular. 2,3 Based on Italian data, the ratio of COVID-related mortality comparing LTCFs residents and people aged over 70 living in the community was estimated to be 3:1. 3 Multiple vaccines against COVID-19 were developed at unprecedented speed, with new vaccine modalities such as mRNAbased vaccines receiving emergency approval. 4,5 Clinical trials report these vaccines are both safe and effective. 6 In Italy, the vaccination campaign against SARS-CoV-2 began in December 2020, with the immunization offered initially to priority groups including healthcare workers (HCWs) and residents of LTCFs. By September 2021, over 90% of LTCF residents had completed a full vaccination cycle. The incidence of weekly SARS-CoV-2 infections among LTCF residents in Italy sharply decreased since the introduction of the vaccine, dropping from around 3.2% new cases per week in November 2020 to 0.01% new cases per week in May-June 2021. The percentage of deceased SARS-CoV-2-positive residents among all LTCF residents also saw an important reduction, and was lower than 0.01% in mid-September 2021. 7 However, clinical trial data on post-vaccine response among elderly and frail individuals is limited. Adaptive immunosenescence, a phenomenon tied to age-related declining immune efficiency, could affect the response to SARS-CoV-2 vaccinations. 4,[8][9][10] This issue is important as older adults are the population at higher risk of developing severe COVID-19. [11][12][13][14] The purpose of this multicentric study was to describe the antibody response to Pfizer/BNT162b2 SARS-CoV-2 mRNA vaccine among individuals at high risk of exposure due to the environment in which they live or work: residents and HCWs of LTCFs. We aimed to provide real-world data from populations, which may have been underrepresented in clinical trials. 2 2 | METHODS

| Study design and participants
This study is part of an ongoing multicenter, prospective cohort study conducted among HCWs (Physicians, Nurses, and Ancillary staff) and residents of 13 LTCFs of the region of Piedmont, in Northern Italy, designed to evaluate SARS-CoV-2 specific IgG titers before and following a complete vaccination cycle with Pfizer/BNT162b2 SARS-CoV-2 mRNA vaccine (two doses of vaccine, 21 days apart). 15 Participants were recruited on a voluntary basis in January 2021, and completed the vaccination cycle between January and March 2021.

| Data collection
Serum samples were obtained from participants at two-time points: (t0) before vaccination, and (t1) 2 weeks after completing a full vaccination cycle. Specimens were processed for cryopreservation as previously described. 15 Demographic characteristics of enrolled subjects, as well as information concerning previous SARS-CoV-2 infections confirmed by reverse-transcription polymerase chain reaction (RT-PCR) testing, were collected from the Health Directorates of the involved facilities and checked on the regional database in which all official swabs are registered. Further, participants were asked whether they had previously been infected by SARS-CoV-2 and if so, when.
In compliance to regional guidelines, from October 2020 all staff and residents of LTCFs are screened for SARS-CoV-2 on a biweekly basis, regardless of symptoms related to COVID-19. Informed consent was obtained before collection of data and specimens.

| Statistical analysis
Demographic and clinical characteristics, including IgG-S titers, were summarized using descriptive statistics. Medians and interquartile ranges (IQRs) were used to describe continuous variables, due to nonnormal distribution (Shapiro-Wilk test), and categorical variables were reported as numbers and percentages. Statistically significant differences in categorical and continuous variables were investigated using χ 2 and Mann-Whitney U tests, respectively.
The Wilcoxon signed-rank test was performed to compare IgG measurements between the first and second blood draws (t0 vs. t1).
Mann-Whitney U tests were conducted to evaluate differences in antibody titers at t1 among HCWs versus residents of LTCFs, and between subjects with and without a previous SARS-CoV-2 infection confirmed by a positive RT-PCR test. The significance level for all analyses was set at two-tailed 0.05. All analyses were conducted using SPSS version 27.0 (IBM).

| RESULTS
Among 952 eligible HCWs and residents of 13 LTCFs, 534 participants were enrolled, and 404 subjects participated in both blood draws. A flowchart of study participants is presented in Figure 1. Table 1 reports demographic and clinical characteristics of participants, stratified according to subject type. HCWs were significantly younger and more often female compared to residents.
A significantly higher proportion of residents had a previous

SARS-CoV-2 infection confirmed by RT-PCR.
Seropositivity among all participants was 50.19% at t0 and 99% at t1. The median IgG titer was 11.34 RU/mL (IQR 0-47.43 RU/mL) at t0 and 1497.28 RU/mL (IQR 779.57-2698.24 RU/mL) at t1 among all subjects. A significant difference in IgG titers at t0 versus t1 was found (p < 0.001 at Wilcoxon signed-rank test). Table 2a,b reports IgG titers among HCWs and residents at t0 and t1, among all participants (Table 2a) and among participants seropositive at t0 (Table 2b). Significant differences were found F I G U R E 1 Study flowchart. Serum samples were obtained from participants at two timepoints: (t0) before vaccination, and (t1) 2 weeks after completing a full vaccination cycle (two doses of vaccine, 21 days apart). Residents had significantly higher titers compared with HCWs at t1 ( Figure 2A). Table 3, significant differences were also found at both time points comparing subjects stratified according to previous SARS-CoV-2 infection confirmed by RT-PCR. At t0, a higher proportion of participants with a previous infection were seropositive compared with participants without a previous infection. IgG titers were significantly higher at t0 in previously infected participants.

As shown in
T A B L E 1 Demographic and clinical characteristics of healthcare workers (HCWs) and residents of 13 long-term care facilities (LTCFs) of the region of Piedmont, in Northern Italy, January 2021 (n = 534). After vaccination, seropositivity in both groups neared 99%, although median IgG titers were almost doubled in previously infected subjects ( Figure 2B). However, as summarized in  Older adults are at increased susceptibility to infections due to immune-senescence, which may also lead to decreased effectiveness of immunizations. 8 even compared with staff. 31 The natural boosting of antibodies due to continued intrafacility transmission could explain results of the current study and our previous findings suggesting a more durable antibody response over time found in residents compared to HCWs of the same LTCFs. 15 T A B L E 3 Differences in immunoglobulin class G (IgG) titers (t0) before and (t1) 2 weeks after receiving two doses of Pfizer/BNT162b2 SARS-CoV-2 mRNA vaccine among healthcare workers (HCWs) and residents of 13 long-term care facilities (LTCFs) of the region of Piedmont, in Northern Italy, stratified according to previous SARS-CoV-2 infection confirmed by RT-PCR, January-March 2021 (n = 534 and 404).  This study had limitations that could affect the generalizability of our findings. First, our cohort consisted of voluntary participants, which may have determined selection bias, and was limited by sample size. Residents lacking the capacity to consent or receiving end-ofcare life were less likely to participate than healthier residents, and other unmeasured differences between participants and individuals declining to participate cannot be excluded. However, we were able to recruit 13 LTCFs varying in size, resident characteristics, and type of care provided. Second, our analysis was restricted by absence of data concerning the timing and clinical characteristics of previous infections, underlying conditions, and frailty status of participants.
Further, even though the same regional screening protocol for SARS-CoV-2 RT-PCR testing was applied in all participating LTCFs since 2020, we cannot exclude that some infections were undiagnosed, as appears to be suggested by results presented in Table 3. Previous analyses suggest surveillance data from the initial stages of the pandemic in Northern Italy were affected by underascertainment. 32 Further, we could not account for other explanations than undetected prior infection for the low-level IgG seen in some participants with no known prior infection, such as cross-reactivity.
Finally, it must also be noted that, as previously discussed, seropositivity may not correlate with protection against reinfection; it is known that the antibodies are only a part of the immune machine, as the role of memory T cells in killing virus-infected cells is equally fundamental. 8,13 It remains to be determined whether infection risk will be different among vaccinated residents and HCWs, and among previously infected versus naïve individuals.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data will be made available upon reasonable request.

ETHICS STATEMENT
The research protocol was in accordance with the Declaration of

TRANSPARENCY STATEMENT
The lead author Valerio Bordino affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.